U.S. patent number 5,683,691 [Application Number 08/462,319] was granted by the patent office on 1997-11-04 for bacillus thuringiensis insecticidal toxins.
This patent grant is currently assigned to Plant Genetic Systems, N.V.. Invention is credited to Henk Joos, Bart Lambert, Marnix Peferoen.
United States Patent |
5,683,691 |
Peferoen , et al. |
November 4, 1997 |
Bacillus thuringiensis insecticidal toxins
Abstract
Two new Bacillus thuringiensis strains, which are deposited at
the DSM under accession nos. 5131 and 5132, produce crystal
proteins during sporulation that are toxic to Coleoptera. The
crystal proteins contain 74 kDa and 129 kDa protoxins,
respectively, which can yield 68 and 66 kDa toxins, respectively,
as trypsin-digestion products. A plant, the genome of which is
transformed with a DNA sequence that comes from either one of the
strains and that codes for its respective toxin, is resistant to
Coleoptera. Each strain, itself, or its crystals, crystal proteins,
protoxin or toxin can be used as the active ingredient in an
insecticidal composition for combatting Coleoptera.
Inventors: |
Peferoen; Marnix (Leuven,
BE), Lambert; Bart (Beernem, BE), Joos;
Henk (Aalter, BE) |
Assignee: |
Plant Genetic Systems, N.V.
(Gent, BE)
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Family
ID: |
27232861 |
Appl.
No.: |
08/462,319 |
Filed: |
June 5, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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247459 |
May 23, 1994 |
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741440 |
Aug 6, 1991 |
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Foreign Application Priority Data
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Feb 15, 1989 [EP] |
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89400428 |
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Current U.S.
Class: |
424/93.461;
435/252.5; 435/832; 530/350; 530/825 |
Current CPC
Class: |
C12N
15/8286 (20130101); Y10S 435/832 (20130101); Y10S
530/825 (20130101); Y02A 40/146 (20180101); Y02A
40/162 (20180101) |
Current International
Class: |
C12N
15/82 (20060101); C12N 001/20 () |
Field of
Search: |
;424/93.461
;435/252.5,832 ;530/350,825 |
References Cited
[Referenced By]
U.S. Patent Documents
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4766203 |
August 1988 |
Kriej et al. |
4771131 |
September 1988 |
Herrnstadt et al. |
4797276 |
January 1989 |
Herrnstadt et al. |
4902507 |
February 1990 |
Morris et al. |
4966765 |
October 1990 |
Payne et al. |
4996155 |
February 1991 |
Sick et al. |
4999192 |
March 1991 |
Payne et al. |
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Foreign Patent Documents
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0193 259 |
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Sep 1986 |
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EP |
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0 213 818 |
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Mar 1987 |
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EP |
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0 289 479 |
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Nov 1988 |
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EP |
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0 328 383 |
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Aug 1989 |
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EP |
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0 337 604 |
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Oct 1989 |
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EP |
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0 342 633 |
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Nov 1989 |
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EP |
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WO 88/08880 |
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Nov 1988 |
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WO |
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WO 89/01515 |
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Feb 1989 |
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WO |
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WO 90/09445 |
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Aug 1990 |
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WO |
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Other References
Sick et al, Nucleic Acids Research, vol. 18, No. 5, p. 1305 (1990).
.
Herrnstadt et al, Bio/Technology, vol. 4, pp. 305-308 (1986). .
Krieg et al, J. Appl. Entomol., vol. 104, No. 4, pp. 417-424
(1987). .
Sekar et al, Proc. Natl. Acad. Sci. USA, vol. 84, No. 29, pp.
7036-7040 (1987). .
Donovan et al, Biosis Database, abstract No. 87-046633
(1987)..
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Primary Examiner: Witz; Jean C.
Attorney, Agent or Firm: Burns, Doane, Swecker & Mathis,
L.L.P.
Parent Case Text
This application is a continuation of application Ser. No.
08/247,459, filed May 23, 1994 now abandoned, which is a
continuation of application Ser. No. 07/741,440, filed Aug. 6,
1991, now abandoned.
Claims
We claim:
1. An isolated BtPGSI245 strain which was deposited under DSM
accession no. 5132 or a strain having all of the identifying
phenotypical characteristics of said BtPGSI245 strain, having
activity against insects of the family Coleoptera.
2. Isolated BtPGSI245 crystals or crystal proteins comprising a
toxin or protoxin having activity against insects of the family
Coleoptera produced by the BtPGSI245 strain of claim 1.
3. Isolated BtPGSI245 protoxin comprising about amino acids 1-1138
of FIG. 2.
4. An insecticidal composition having activity against Coleoptera,
which comprises the isolated BtPGSI245 strain of claim 1.
5. A process for controlling a Coleopteran pest comprising
contacting said pest with an insecticidally effective amount of the
insecticidal composition of claim 4.
6. The process of claim 5 wherein said Coleopteran pest is
Leptinotarsa decemlineata, Agelastica alni, Diabrotica luteola,
Haltica tombacina, Anthonomus grandis, Tenebrio molitor, Diabrotica
undecimpunctata or Triboleum castaneum.
7. The process of claim 6 wherein the pest is Leptinotarsa
decemlineata.
8. An insecticidal composition having activity against Coleoptera
which comprises the isolated BtPGSI245 crystals or crystal proteins
of claim 2.
9. An insecticidal composition having activity against Coleoptera
which comprises the isolated BtPGSI245 protoxin of claim 3.
10. Isolated BtPGSI245 toxin comprising about amino acids 53-637 of
FIG. 2.
11. An insecticidal composition having activity against Coleoptera
which comprises the isolated BtPGSI245 toxin of claim 10.
12. A process for controlling a Coleopteran pest comprising
contacting said pest with an insecticidally effective amount of the
insecticidal composition of claim 8.
13. A process for controlling a Coleopteran pest comprising
contacting said pest with an insecticidally effective amount of the
insecticidal composition of claim 9.
14. A process for controlling a Coleopteran pest comprising
contacting said pest with an insecticidally effective amount of the
insecticidal composition of claim 11.
Description
This invention relates to two new strains of B. thuringiensis (the
"BtPGSI208 strain" and the "BtPGSI245 strain"), each of which
produces crystallized proteins (the "BtPGSI208 crystal proteins"
and the "BtPGSI245 crystal proteins", respectively) which are
packaged in crystals (the "BtPGSI208 crystals" and the "BtPGSI245
crystals", respectively) during sporulation. The BtPGSI208 and
BtPGSI245 strains were deposited under the provisions of the
Budapest Treaty at the Deutsche Sammlung Fur Mikroorganismen and
Zellkulturen ("DSM"), Mascheroder Weg 1B, D-3300 Braunschweig,
Federal Republic of Germany, under accession numbers 5131 and 5132,
respectively, on Jan. 19, 1989.
This invention also relates to an insecticide composition that is
active against Coleoptera and that comprises the BtPGSI208 or
BtPGSI245 strain, as such, or preferably the BtPGSI208 or BtPGSI245
crystals, crystal proteins or the active component(s)
ingredient.
This invention further relates to:
1) a DNA sequence (the "btPGSI208 gene"), from the genome of the
BtPGSI208 strain, which encodes a 74 kDa protein (the "BtPGSI208
protoxin") that is found in the BtPGSI208 crystals; and
2) A DNA sequence (the "btPGSI245 gene), from the genome of the
BtPGSI245 strain, which encodes a 129 kDa protein (the "BtPGSI245
protoxin") that is found in the BtPGSI245 crystals.
The BtPGSI208 and BtPGSI245 protoxins are the proteins that are
produced by their respective BtPGSI208 and BtPGSI245, strains
before being packaged into their respective BtPGSI208 and BtPGSI245
crystals.
This invention still further relates to a 68 kDa protein ("the
BtPGSI208 toxin") and a 66 kDa protein (the "BtPGSI245 toxin")
which can be obtained (e.g., by trypsin digestion) from the
BtPGSI208 protoxin and the BtPGSI245 protoxin, respectively. The
BtPGSI208 and BtPGSI245 toxins are insecticidally active proteins
which can be liberated from the BtPGSI208 crystals and the
BtPGSI245 crystals, respectively, produced by the BtPGSI208 strain
and the BtPGSI245 strain, respectively, and each toxin has a high
activity against Coleoptera. The BtPGSI208 and BtPGSI245 toxins are
believed to represent the smallest portions of their respective
BtPGSI208 and BtPGSI245 protoxins which are insecticidally
effective against Coleoptera.
This invention yet further relates to a chimaeric gene that can be
used to transform a plant cell and that contains:
1) a part of the btPGSI208 or btPGSI245 gene (the "insecticidally
effective btPGSI208 or btPGSI245 gene part") encoding an
insectidicidally effective portion of the respective BtPGSI208 or
BtPGSI245 protoxin, preferably a truncated part of the btPGSI208 or
btPGSI245 gene (the "truncated btPGSI208 or btPGSI245 gene")
encoding just the respective BtPGSI208 or BtPGSI245 toxin;
2) a promoter suitable for transcription of the insecticidally
effective btPGSI208 or btPGSI245 gene part in a plant cell; and
3 ) suitable transcription termination and polyadenylation signals
for expressing the insecticidally effective btPGSI208 or btPGSI245
gene part in a plant cell.
This chimaeric gene is hereinafter generally referred to as the
"btPGSI208 or btPGSI245 chimaeric gene." Preferably, the
insecticidally effective btPGSI208 or btPGSI245 gene part is
present in the btPGSI208 or btPGSI245 chimaeric gene as a hybrid
gene comprising a fusion of the truncated btPGSI208 or btPGSI245
gene and a selectable marker gene, such as the neo gene (the
"btPGSI208-neo or btPGSI245-neo hybrid gene") encoding a
BtPGSI208-NPTII or BtPGSI245-NPTII fusion protein.
This invention also relates to:
1) a cell (the "transformed plant cell") of a plant, such as
potato, the genome of which is transformed with the insecticidally
effective btPGSI208 or btPGSI245 gene part; and
2) a plant (the "transformed plant") which is regenerated from the
transformed plant cell or is produced from the so-regenerated
plant, the genome of which contains the insecticidally effective
btPGSI208 or btPGSI245 gene part and which is resistant to
Coleoptera.
This invention still further relates to a B. thuringiensis ("Bt")
strain transformed, preferably by electroporation, with a vector
carrying all or part of the btPGSI208 or btPGSI245 gene.
BACKGROUND OF THE INVENTION
B. thuringiensis is a gram-positive bacterium which produces
endogenous crystals upon sporulation. The crystals are composed of
proteins which are specifically toxic against insect larvae. Three
different Bt pathotypes have been described: pathotype A that is
active against Lepidoptera, e.g., caterpillars; pathotype B that is
active against certain Diptera, e.g., mosquitos and black flies;
and pathotype C that is active against Coleoptera, e.g., beetles
(Ellar et al, 1986).
A Bt strain, whose crystals are toxic to Coleoptera, has been
described as Bt tenebrionis (U.S. Pat. No. 4,766,203; European
patent publication 0,149,162), Bt M-7 or Bt San Diego (European
patent publication 0,213,818; U.S. Pat. No. 4,771,131) and BtS1
(European patent application 88/402,115.5).
The fact that conventional submerged fermentation techniques can be
used to produce Bt spores on a large scale makes Bt bacteria
commercially attractive as a source of insecticidal
compositions.
Gene fragments from some Bt strains, encoding insecticidal
proteins, have heretofore been identified and integrated into plant
genomes in order to render the plants insect-resistant. However,
obtaining expression of such Bt gene fragments in plants is not a
straightforward process. To achieve optimal expression of an
insecticidal protein in plant cells, it has been found necessary to
engineer each Bt gene fragment in a specific way so that it encodes
a water-soluble part of a Bt protoxin that retains substantial
toxicity against its target insects (European patent applications
86/300,291.1 and 88/402,115.5; U.S. patent application Ser. No.
821,582, filed Jan. 22, 1986)
SUMMARY OF THE INVENTION
In accordance with this invention, the two new Bt strains of
pathotype C, i.e., the BtPGSI208 and BtPGSI245 strains, are
provided. The BtPGSI208 and BtPGSI245 crystals, crystal proteins,
protoxins and toxins, produced by the respective strains during
sporulation, as well as insecticidally effective portions of the
BtPGSI208 and BtPGSI245 protoxins, each possess insecticidal
activity and can therefore be formulated into insecticidal
compositions against Coleoptera in general, especially against
Agelastica alni, Diabrotica luteola, Haltica tombacina, Anthonomus
grandis, Tenebrio molitor, Diabrotica undecimpunctata and Triboleum
castaneum and particularly against the Colorado potato beetle,
Leptinotarsa decemlineata, which is a major pest of economically
important crops.
Also in accordance with this invention, a plant cell genome is
transformed with the insecticidally effective btPGSI208 or
btPGSI245 gene part, preferably the truncated btPGSI208 or
btPGSI245 gene. It is preferred that this transformation be carried
with the btPGSI208 or btPGSI245 chimaeric gene. The resulting
transformed plant cell can be used to produce a transformed plant
in which the plant cells in some or all of the plant tissues: 1)
contain the insecticidally effective btPGSI208 or btPGSI245 gene
part as a stable insert in their genome and 2) express the
insecticidally effective btPGSI208 or btPGSI245 gene part by
producing an insecticidally effective portion of its respective
BtPGSI208 or BtPGSI245 protoxin, preferably its respective
BtPGSI208 or BtPGSI245 toxin, thereby rendering the plant resistant
to Coleoptera. The transformed plant cells of this invention can
also be used to produce, for recovery, such insecticidal Bt
proteins.
Further in accordance with this invention, a process is provided
for rendering a plant resistant to Coleoptera by transforming the
plant cell genome with the insecticidally effective btPGSI208 or
btPGSI245 gene part, preferably the truncated btPGSI208 or
btPGSI245 gene. In this regard, it is preferred that the plant cell
be transformed with the btPGSI208 or btPGSI245 chimaeric gene.
Still further in accordance with this invention, there are provided
the BtPGSI208 and BtPGSI245 protoxins, the insecticidally effective
portions of such protoxins and the BtPGSI208 and BtPGSI245 toxins,
as well as the btPGSI208 and btPGSI245 genes, the insecticidally
effective btPGSI208 and btPGSI245 gene parts, the truncated
btPGSI208 and btPGSI245 genes and the chimaeric btPGSI208 and
btPGSI245 genes.
Yet further in accordance with this invention, a Bt strain is
transformed, preferably by electroporation, with a vector carrying
all or part of the btPGSI208 or btPGSI245 gene encoding all or an
insecticidally effective portion of the BtPGSI208 or BtPGSI245
protoxin.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with this invention, the BtPGSI208 and BtPGSI245
protoxins can be isolated in a conventional manner from,
respectively, the BtPGSI208 strain, deposited at the DSM under
accession number 5131, and the BtPGSI245 strain, deposited at the
DSM under accession number 5132. For example, the BtPGSI208 and
BtPGSI245 crystals can be isolated from sporulated cultures of
their respective strains (Mahillon and Delcour, 1984), and then,
the respective protoxins can be isolated from these crystals
according to the method of Hofte et al (1986). The protoxins can be
used to prepare monoclonal or polyclonal antibodies specific for
these protoxins in a conventional manner (Hofte et al, 1988). The
BtPGSI208 toxin can then be obtained by removing (e.g., by trypsin
digestion) approximately 57 N-terminal amino acids from the
BtPGSI208 protoxin. The BtPGSI245 toxin can be obtained by removing
(e.g., by trypsin digestion) approximately 52 N-terminal and
approximately 501 C-terminal amino acids from the BtPGSI245
protoxin.
The btPGSI208 and btPGSI245 genes can also be isolated from their
respective strains in a conventional manner. For example, the
btPGSI208 or btPGSI245 gene can be identified in its respective
BtPGSI208 or BtPGSI245 strain, using the procedure described in
U.S. patent application Ser. No. 821,582, filed Jan. 22, 1986, and
in European patent applications 86/300,291.1 and 88/402,115.5
(which are incorporated herein by reference). Preferably, the
btPGSI208 and btPGSI245 genes are each identified by: digesting
total DNA from their respective BtPGSI208 and BtPGSI245 strains
with one or more restriction enzymes; size fractionating the DNA
fragments, so produced, into DNA fractions of 5 to 10 Kb; ligating
such fractions to cloning vectors; transforming E. coli with the
cloning vectors; and screening the clones with a suitable DNA
probe. The DNA probe can be constructed: 1) from a highly conserved
region of a Bt gene which codes for another crystal protoxin
against Coleoptera such as: the bt13 gene described in European
patent application 88/402,115.5 and by Hofte et al (1987); or 2) on
the basis of the N-terminal amino acid sequence of the protoxin
encoded by the respective btPGSI208 or btPGSI245 gene, which
sequence can be determined by gas-phase sequencing of the
immobilized protoxin (European Patent application
88/402,115.5).
Alternatively, the 5 to 10 kB fragments, prepared from total DNA of
the BtPGSI208 or BtPGSI245 strain, can be ligated in suitable
expression vectors and transformed in E. coli, and the clones can
then be screened by conventional colony immunoprobing methods
(French et al, 1986) for expression of the BtPGSI208 or BtPGSI245
toxin with monoclonal or polyclonal antibodies raised against the
toxin.
The so-identifed btPGSI208 and btPGSI245 genes can then each be
sequenced in a conventional manner (Maxam and Gilbert, 1980) to
obtain the DNA sequences shown in FIGS. 1 and 2, respectively. The
nucleotide sequences of the btPGSI208 gene and btPGSI245 gene,
shown in FIGS. 1 and 2, prove that the BtPGSI208 and BtPGSI245
protoxins and toxins are different from previously described
protoxins and toxins with activity against Coleoptera (Hofte and
Whiteley, 1989).
An insecticidally effective part of each of the sequenced genes,
encoding an insecticidally effective portion of its protoxin, and a
truncated part of each of the sequenced genes, encoding just its
toxin, can be made in a conventional manner from each gene after
the gene has been sequenced. The aminoacid sequences of the
BtPGSI208 and BtPGSI245 protoxins and toxins can be determined from
the DNA sequences of their respective btPGSI208 and btPGSI245 genes
and truncated btPGSI208 and btPGSI245 genes. By "an insecticidally
effective part" or "a part" of the btPGSI208 or btPGSI245 gene is
meant a DNA sequence encoding a polypeptide which has fewer amino
acids then the respective BtPGSI208 or BtPGSI245 protoxin but which
is still toxic to Coleoptera. Such a part of the btPGSI208 or
btPGSI245 gene can encode a BtPGSI208 or BtPGSI245 protoxin which
has been truncated towards at least one trypsin cleavage site of
the protoxin (U.S. patent application Ser. No. 821,582; European
patent application 86/300291.1).
In order to express all or an insecticidally effective part of the
btPGSI208 or btPGSI245 gene in E. coli and in plants, suitable
restriction sites are introduced, flanking each gene or gene part.
This can be done by site directed mutagenesis, using well-known
procedures (Stanssens et al, 1987; Stanssens et al, 1989).
The insecticidally effective btPGSI208 or btPGSI245 gene part,
encoding an insecticidally effective portion of its respective
BtPGSI208 or BtPGSI245 protoxin, can be stably inserted in a
conventional manner into the nuclear genome of a single plant cell,
and the so-transformed plant cell be used in a conventional manner
to produce a transformed plant that is insect-resistant. In this
regard, a disarmed Ti-plasmid, containing the insectidicidally
effective btPGSI208 or btPGSI245 gene part, in Agrobacterium
tumefaciens can be used to transform the plant cell, and
thereafter, a transformed plant can be regenerated from the
transformed plant cell using the procedures described, for example,
in European patent publications 0,116,718 and 0,270,822, PCT
publication WO 84/02,913 and European patent application
87/400,544.0 (which are also incorporated herein by reference).
The resulting transformed plant can be used in a conventional plant
breeding scheme to produce more transformed plants with the same
characteristics or to introduce the insecticidally effective
btPGSI208 or btPGSI245 gene part in other varieties of the same or
related plant species. Seeds, which are obtained from the
transformed plants, contain the insecticidally effective btPGSI208
or btPGSI245 gene part as a stable genomic insert. Cells of the
transformed plant can be cultured in a conventional manner to
produce the BtPGSI208 or BtPGSI245 protoxin, preferably the
respective toxin, which can be recovered for use in conventional
insecticide compositions against Coleoptera (U.S. patent
application Ser. No. 821,582; European patent application
86/300291.1.).
The insecticidally effective btPGSI208 or btPGSI245 gene part,
preferably the truncated btPGSI208 or btPGSI245 gene, is inserted
in a plant cell genome so that the inserted part of the gene is
downstream (i.e., 3') of, and under the control of, a promoter
which can direct the expression of the gene part in the plant cell.
This is preferably accomplished by inserting the btPGSI208 or
btPGSI245 chimaeric gene in the plant cell genome. Preferred
promoters include: the strong constitutive 35S promoters (the "35S
promoters") of the cauliflower mosaic virus of isolates CM 1841
(Gardner et al, 1981), CabbB-S (Franck et al, 1980) and CabbB-JI
(Hull and Howell, 1987); and the TR1' promoter and the TR2'
promoter (the "TR1' promoter" and "TR2' promoter", respectively)
which drive the expression of the 1' and 2' genes, respectively, of
the T-DNA (Velten et al, 1984). Alternatively, a promoter can be
utilized which is not constitutive but rather is specific for one
or more tissues or organs of the plant (e.g., leaves and/or roots)
whereby the inserted btPGSI208 or btPGSI245 gene part is expressed
only in cells of the specific tissue(s) or organ(s). For example,
the btPGSI208 or btPGSI245 gene part could be selectively expressed
in the leaves of a plant (e.g., potato) by placing the gene part
under the control of a light-inducible promoter such as the
promoter of the ribulose-1,5-bisphosphate carboxylase small subunit
gene of the plant itself or of another plant such as pea as
disclosed in U.S. patent application Ser. No. 821,582 and European
patent application 86/300,291.1. Another alternative is to use a
promoter whose expression is inducible (e.g., by temperature or
chemical factors).
The insecticidally effective btPGSI208 or btPGSI245 gene part is
inserted in the plant genome so that the inserted part of the gene
is upstream (i.e., 5') of suitable 3' transcription regulation
signals (i.e., transcription termination and polyadenylation
signals). This is preferably accomplished by inserting the
btPGSI208 or btPGSI245 chimaeric gene in the plant cell genome.
Preferred polyadenylation and transcription termination signals
include those of the octopine synthase gene (Gielen et al, 1984)
and the T-DNA gene 7 (Velten and Schell, 1985), which act as
3'-untranslated DNA sequences in transformed plant cells.
It is preferred that the insecticidally effective btPGSI208 or
btPGSI245 gene part be inserted in the plant genome in the same
transcriptional unit as, and under the control of, the same
promoter as a selectable marker gene. The resulting hybrid
btPGSI208 or btPGSI245-marker gene will, thereby, be expressed in a
transformed plant as a fusion protein (U.S. patent application Ser.
No. 821,582; European patent application 86/300291.1; Vaeck et al,
1987). This result is preferably accomplished by inserting a
btPGSI208 or btPGSI245 chimaeric gene, containing the marker gene,
in the plant cell genome. Any conventional marker gene can be
utilized, the expression of which can be used to select transformed
plant cells. An example of a suitable selectable marker gene is an
antibiotic resistance gene such as the neo gene coding for
kanamycin resistance (Reiss et al, 1984; European patent
application 87/400,544.0; U.S. patent application Ser. No. 821,582;
European patent application 86/300,291.1). Thereby, the
insecticidally effective btPGSI208 or btPGSI245 gene part and the
marker gene (e.g. the btPGSI208-neo or btPGSI245-neo hybrid gene)
are expressed in a transformed plant as a fusion protein (U.S.
patent application Ser. No. 821,582; European patent application
86/300,291.1; Vaeck et al, 1987).
All or part of the btPGSI208 and btPGSI245 genes, encoding
Coleopteran toxins, can also be used to transform gram-positive
bacteria, such as a B. thuringiensis which has insecticidal
activity against Lepidoptera or Coleoptera. Thereby, a transformed
Bt strain can be produced which useful for combatting both
Lepidopteran and Coleopteran insect pests or combatting additional
Coleopteran insect pests. Transformation of a bacteria with all or
part of the btPGSI208 or btPGSI245 gene, incorporated in a suitable
cloning vehicle, can be carried out in a conventional manner,
preferably using conventional electroporation techniques as
described in PCT patent application PCT/EP89/01539, filed Dec. 11,
1989.
Each of the BtPGSI208 and BtPGSI245 strains can be fermented by
conventional methods (Dulmage, 1981) to provide high yields of
cells. Under appropriate conditions which are well understood
(Dulmage, 1981), the BtPGSI208 and BtPGSI245 strains each sporulate
to provide their respective BtPGSI208 and BtPGSI245 crystal
proteins in high yields.
An insecticide composition of this invention can be formulated in a
conventional manner using the BtPGSI208 or BtPGSI245 strain or
preferably their respective crystals, crystal proteins, protoxin,
toxin and/or insecticidally effective portions of their respective
protoxin as active ingredient(s), together with suitable carriers,
diluents, emulsifiers and/or dispersants. This insecticide
composition can be formulated as a wettable powder, pellets,
granules or a dust or as a liquid formulation with aqueous or
non-aqueous solvents as a foam, gel, suspension, concentrate, etc.
The concentration of the BtPGSI208 or BtPGSI245 strain, crystals,
crystal proteins, protoxin, toxin and/or protoxin portions in such
a composition will depend upon the nature of the formulation and
its intended mode of use. Generally, an insecticide composition of
this invention can be used to protect a potato field for 2 to 4
weeks against Coleoptera with each application of the composition.
For more extended protection (e.g., for a whole growing season),
additional amounts of the composition should be applied
periodically.
BRIEF DESCRIPTION OF THE DRAWINGS
The following Examples illustrate the invention. The figures,
referred to in the Examples, are as follows:
FIG. 1--DNA sequence of the btPGSI208 gene. The derived aminoacid
sequence of the encoded BtPGSI208 protoxin is presented beneath
this sequence. The arrow separates the N-terminal 57 aminoacids
from the C-terminal portions encoding the BtPGSI208 toxin. The
truncated btPGSI208 gene, coding just for the BtPGSI208 toxin,
extends from nucleotide position 513 (see arrow) to the TAG
termination codon at nucleotide position 2295.
FIG. 2--DNA sequence of the btPGSI245 gene. The derived aminoacid
sequence of the encoded BtPGSI245 protoxin is presented beneath
this sequence. The arrows delineate the BtPGSI245 toxin between
aminoacids 52 and 638 of the BtPGSI245 protoxin. The truncated
btPGSI245 gene, coding just for the BtPGSI245 gene, extends from
nucleotide position 340 (see arrow) to nucleotide position 2094
(see arrow).
FIG. 3--Total protein patterns by SDS-PAGE of sporulated BtPGSI208
and BtPGSI245 and other Bacillus cultures. Among the comparison
strains, B. subt. is Bacillus subtilis, B. cer. is Bacillus cereus,
and Bt Darm is Bacillus thuringiensis subsp. darmstadiensis. These
comparison strains were obtained from the sources set forth in
Table 1, hereinafter. "MW" designates molecular weight markers.
FIG. 4A--Protein blotting of total proteins and trypsinized crystal
proteins from strains BtS1 and BtPGSI208. Total protein patterns
were stained with Indian ink, while crystal proteins were
visualized with an antiserum against Bt13 toxin ("anti-CryIIIA").
"HMW" designates molecular weight markers.
FIG. 4B--Protein blotting of total proteins and trypsinized crystal
proteins from strains BtS1, BtPGSI245 and Bt HD-110. Total protein
patterns were probed for their immunoreactivity with an antiserum
against Bt13 toxin ("anti-CryIIIA") and an antiserum against Bt2
protoxin ("anti-CryIA(b)"). "LMW" designates molecular weight
markers. The comparison strain, HD-110, was Bt HD-110, obtained
from Dr. H. Dulmage, Cotton Insect Laboratories, U.S.D.A.,
Brownsville, Texas, U.S.A.
Unless otherwise stated in the Examples, all procedures for making
and manipulating recombinant DNA are carried out by the
standardized procedures described in Maniatis et al, Molecular
Cloning--A laboratory Manual, Cold Spring Harbor Laboratory
(1982).
EXAMPLE 1
Characterization of the BtPGSI208 and BtPGSI245 strains
The BtPGSI208 strain was isolated from grain dust sampled in
Belgium and was deposited at the DSM on Jan. 19, 1989 under
accession No. 5131.
The BtPGSI245 strain was isolated from cow dung sampled in the
United States and was deposited at the DSM on Jan. 19, 1989 under
accession No. 5132.
Each strain can be cultivated on conventional standard media,
preferably LB medium (Bacto-tryptone 10 g/l, yeast extract 5 g/l,
NaCl 10 g/l and agar 15 g/l), preferably at 28.degree. C. For long
term storage, it is preferred to use LB liquid medium containing
50% glycerol at -70.degree. C. or lyophilization. For sporulation,
the use of T.sub.3 medium (tryptone 3 g/l, tryprose 2 g/l, yeast
extract 1.5 g/l, 5 mg MnCl.sub.2, 0.05M Na.sub.2 PO.sub.4, pH 6.8
and 1.5% agar) is preferred for 24 hours at 28.degree. C., followed
by storage at 4.degree. C. During its vegetative phase, each of the
BtPGSI208 and BtPGSI245 strains can also grow under facultative
anaerobic conditions, but sporulation only occurs under aerobic
conditions.
Sterilization of each strain occurs by autoclave treatment at
120.degree. C. (1 bar pressure) for 20 minutes. Such treatment
totally inactivates the spores and the crystalline BtPGSI208 and
BtPGSI245 protoxins. UV radiation (254 nm) inactivates the spores
but not the protoxins.
After cultivating on Nutrient Agar ("NA", Difco Laboratories,
Detroit, Mich., U.S.A.) for one day, colonies of each of the
BtPGSI208 and BtPGSI245 strains form opaque white colonies with
irregular edges. Cells of each strain (Gram positive rods of
1.7-2.4.times.5.6-7.7 .mu.m) sporulate after three days cultivation
at 28.degree. C. on NA. The crystal proteins produced during
sporulation are packaged in flat rhomboid crystals in the BtPGSI208
strain and in bipyramidal crystals in the BtPGSI245 strain.
For the biochemical characterization of the two strains, the
following tests were carried out using well known methods as
described for example by Sheath et al (1986). Growth was observed
in Nutrient Broth ("NB", Difco) supplemented with 2 and 5% NaCl. No
growth of the BtPGSI208 strain and only weak growth of the
BtPGSI245 strain were observed in the presence of 7% NaCl. Neither
strain grew in medium supplemented with 10% NaCl. The BtPGSI208 and
BtPGSI245 strains grew well on NA at 20, 28 and 37.degree. C., but
not at 4, 10 (although the BtPGSI245 strain grew slowly at this
temperature), 50.degree. and 60.degree. C. Both strains grew in NB
at pH=5, pH=6 and pH=7 and on NB containing 100 units of lysozyme
(Sigma Chemical Company, St Louis, Mo., U.S.A.) per ml of NB.
Growth on NA under anaerobiosis was very weak.
Metabolic characteristics of the two strains were determined using
API-20E test strips (API Systems S.A., Montalieu-Vercieu, France).
The results of these assays are shown in Table 1, below.
TABLE 1 ______________________________________ Metabolic
characteristics of the BtPGSI208 and BtPGSI245 strains as compared
with other Bacillus strains (+ = positive reaction; - = negative
reaction; w = weak reaction; nd = not determined). Bt Bt PGSI PGSI
Activity 208 245 BTS1 BTEN BDAR BCER BSUB
______________________________________ ONPG - - - - - - + ADH - + +
+ + + - LDC - - - - - - - ODC - - - - - - - CIT - - - - - - - H2S -
- - - - - - URE - - - - - - - TDA - - - + w + + IND - - - - - - -
VP - - - w w w + GEL - + - + + + + OX + + + + + + + NO2 + + + + + +
nd N2 - - - - - - nd ______________________________________ ONPG =
pgalactosidase activity. ADH = arginine dihydrolase activity. LDC =
lysine decar oxylase activity. ODC = ornithine decarboxylase
activity. CIT = use of citrate as sole carbon source. H2S = H.sub.2
S formation from thiosulphate. URE = urease activity. TDA =
tryptophan deaminase activity. IND = indol formation from
tryptophan. VP = acetoin formation from sodium pyruvate. GEL =
gelatin liquefaction. OX = moxidase activity. NO2 = nitrate
reduction to nitrite. N2 = N.sub.2 gas production from nitrate.
BTS1 = Bacillus thuringiensis BtS1 from DSM under accession no.
4288. BTEN = Bacillus thuringiensis subsp. tenebrionis from DSM
under accession no. 2803. BDAR = Bacillus thuringiensis subsp.
darmstadiensis from Institut fur Landwirtschaftliche Bacteriologie
und Garungsbiologie der Eidgenossiche Technische Hochschule,
Zurich, Switzerland ("LBG"), under accession no. 4447 BCER =
Bacillus cereus from Laboratorium voor Microbiologie, Gent, Belgiu
("LMG"), under accession no. 2098. BSUB = Bacillus subtillis from
Agricultural Research Culture Collection, Peoria, Illinois, USA,
under accession no. NRRL B237.
Both strains were found to rapidly decompose casein in skim-milk
agar and to deaminate phenylalanine in tests described by Sheath et
al (1986).
Acid production from different sugars by the two strains was
determined using API-50CHB test strips (API Systems SA). The
results are shown in Table 2, below.
TABLE 2 ______________________________________ Acid production by
the BtPGSI208 and BtPGSI245 strains as compared with other bacilli
(+ = positive reaction; - = negative reaction; w = weak reaction).
Bt Bt PGSI PGSI Substrate: 208 245 BTS1 BTEN BDAR BCER BSUB
______________________________________ Control - - - - - - -
Glycerol - w + + + + + Erythritol - - - - - - - D-arabinose - - - -
- - - L-arabinose - - - - - - + Ribose + + + + + + + D-Xylose - - -
- - - + L-Xylose - - - - - - - Adonitol - - - - - - - B Methyl- - -
- - - - - xyloside Galactose - - - - - - + D-Glucose + + + + + + +
D-Fructose + + + + + + + D-Mannose - + + + - - + L-Sorbose - - - -
- - - Rhamnose - - - - - - - Dulcitol - - - - - - - Inositol - - -
- - - + Mannitol - - - - - - + Sorbitol - - - - - - +
.alpha.-Methyl-D- - - - - - - - mannoside .alpha.-Methyl-D- - - - -
- - + qlucoside N-Acetyl- + + + + + + - glucosamide Amygdaline - -
- - - - + Arbutine + + + + + - + Esculine + + + + + + + Salicine +
w - - - - + Cellobiose - w - - - - + Maltose + + + + + + + Lactose
- - - - - - + Melibiose - - - - - - + Saccharose + + + + - + +
Trehalose + + + + + + + Inuline - - - - - - + Melizitose - - - - -
- - D-Raffinose - - - - - - + Starch + + + + + + + Glycogen + + + +
+ + + Xylitol - - - - - - - B Gentio- - - - - - - - biose
D-Turanose - - - - - - + D-Lyxose - - - - - - - D-Tagatose - - - -
- - - D-Fucose - - - - - - - L-Fucose - - - - - - - D-Arabitol - -
- - - - - L-Arabitol - - - - - - - Gluconate - - - - - - - 2 Keto-
- - - - - - - gluconate 5 Keto- - - - - - - - gluconate
______________________________________
Sensitivity of the two strains towards different antibiotics was
tested using Oxoid Susceptibility Test Discs on Oxoid Isosensitest
agar ("CM471" of Oxoid Ltd., Basingstoke, Hampshire, England). The
results are shown in Table 3, below.
TABLE 3
__________________________________________________________________________
Antibiotic sensitivity as shown by the diameters (in mm) of
inhibition zones observed after 24 hours on antibiotic-containing
agar, seeded with different bacilli (R = resistant colonies or no
growth detected). amount/ Bt PGSI Bt PGSI Antibiotic disc 208 245
BTS1 BTEN BDAR BCER BSUB
__________________________________________________________________________
Chloramphenicol 30 ug 25/R 17 19/R 20 22 28 33 Bacitracin 10 i.u 11
10 8 7 14 18 7 Gentamycin 10 ug 26 20 21 20 28 9 25/R Neomycin 30
ug 24 20/R 13/R 13 26 10 20 Tetracyclin 30 ug 14/R 10 17/R 16/R
10/R 21 22 Carbenicillin 100 ug 8 11 0 0 10 0 19 Rifampicin 2 ug 12
13 0 8 8 19 26 Penicillin G 10 i.u 7 8 0 0 0 14/R 14 Streptomycin
10 ug 25/R 15 16 17 20 14 0 Spectinomycin 10 ug 0 0 0 0 0 12/R 0
Kanamycin 30 ug 20/R 21/R 0 0 24 15 24 Nalidixic acid 30 ug 25/R
23/R 18/R 25/R 30/R 7 19 Sulphamethoxazole 25 ug 0 0 0 0 0 0 0
Trimethoprin 2.5 ug 0 0 0 0 0 0 26 Ampicillin 10 u 7 9/R 0 0 0 15/R
18
__________________________________________________________________________
The enzyme spectra of the BtPGSI208 and BtPGSI245 strains were
determined using the extended API-ZYM strips (API Systems S.A.).
The results are shown in Table 4, below. Esterass-, peptidase-
(AP1, AP2, AP3, AP4, AP5 and AP6 test strips) and osidase-test
strips were inoculated with 50 ul cell suspension (10.sup.7
cfu/ml). The osidase reaction was revealed after 4 hours incubation
(28.degree. C.) with 25 .mu.l 0.1N NaOH. All other reactions were
with 25 .mu.l ZYM A and ZYM B reagent (API no. 7048).
TABLE 4 ______________________________________ Enzymatic spectra of
the BtPGSI208 and BtPGSI245 strains as compared to two other Bt
strains (0 = no substrate used; 1. 2, 3, 4, 5 = 5, 10, 20, 30 and
.gtoreq.40 nanomoles of substrate hydrolysed respectively). Bt Bt
PGSI PGSI Substrate 208 245 BTS1 BDAR
______________________________________ Esterases.
2-naphtyl-valerate 4 4 2 4 2-naphtyl-caproate 5 5 5 5
2-naphtyl-caprylate 5 5 5 5 2-naphtyl-nonanoate 5 5 4 5
2-naphtyl-caprate 5 3 3 5 2-naphtyl-laurate 2 2 1 2
2-naphtyl-myristate 1 2 1 1 2-naphtyl-palmitate 0 0 1 0
2-naphtyl-stearate 2 1 2 2 Peptidases.
L-pyrrolidonyl-.beta.-naphtylamide 0 5 5 5
Glycyl-.beta.-naphtylamide 0 0 0 0 L-glutamyl-.beta.-naphtylamide 0
0 0 0 L-leucyl-glycyl-.beta.-naphtylamide 0 1 0 0
L-seryl-L-tyrosyl-.beta.-naphtylamide 4 5 5 5
L-glutamine-.beta.-naphtylamide 1 5 4 5
L-glutanyl-.beta.-naphtylamide 0 3 3 3 Osidases.
Paranitrophenol-D-galactopyrano- 0 0 0 0 side
Paranitrophenol-.beta.D-galactopyra- 0 0 0 0 noside
Paranitrophanol-aD-glucopyrano- 5 5 5 5 side
Paranitrophenol-.beta.D-glucopyrano- 0 2 0 0 side
Paranitrophenol-a-maltoside 2 4 3 5
Paranitrophenol-.beta.-maltoside 0 0 0 0 Paranitrophenol-
N-acetyl-.beta.D-glucosamidine 3 5 5 5
Paranitrophenol-.beta.D-xylapyrano- 0 0 0 0 side
______________________________________
EXAMPLE 2
Characteristics of the BtPGSI208 and BtPGSI245 crystals
The BtPGSI208 and BtPGSI245 strains were grown for 48 to 72 hours
at 28.degree. C. on T.sub.3 medium. After sporulation, the spores
and crystals were harvested in phosphate buffered saline solution
("PBS" from Oxoid Ltd.) by scraping with a Trihalski spatula. The
resulting aqueous spore-crystal suspensions were centrifuged, and
the pellets were resuspended and incubated overnight in aqueous
solutions containing 50 mM Na.sub.2 CO.sub.3 and 5 mM dithiotreitol
("DTT") at pH 10. After centrifugation, the supernatants were
recovered containing the respective crystal proteins.
The BtPGSI208 protoxin and toxin, as well as the BtPGSI245 toxin,
react only with a polyclonal antiserum raised against the Bt13
toxin as shown in FIGS. 4A & 4B. In contrast, the BtPGSI245
protoxin reacts with polyclonal antisera against the BtS1 or Bt13
toxin (European patent application 88/402115.5) and the Bt2
protoxin (U.S. patent application 821,582; European patent
application 86/300,291.1) as shown in FIGS. 4A and 4B.
The total protein patterns of the BtPGSI208 and BtPGSI245 strains,
compared to other Bacillus strains, are shown in FIG. 3. For this
comparison, the crystal proteins of each strain were analyzed on a
12.5% SDS-PAGE gel (Laemmli, 1970) and stained with Coomassie
brilliant blue R-250 according to Lambert et al (1987). The crystal
proteins were dissolved by exposing the spore-crystal mixtures
overnight at 37.degree. C. to 50 mM Na.sub.2 CO.sub.3, pH 10, 5 mM
DTT. Solubilized crystal proteins were digested by adjusting the pH
to 9.0 with 0.5M HCl and by trypsinization (1 .mu.g bovine
trypsin/25 .mu.g protein). Trypsin digestion of the BtPGSI208 and
BtPGSI245 crystal proteins was performed at 37.degree. C. overnight
and revealed the presence of tryptic fragments of 68 kDa and 66
kDa, respectively (FIGS. 4A and 4B). Immunoblotting experiments,
performed according to Peferoen (1988) with polyclonal antisera
raised against the Bt13 toxin and the Bt2 protoxin demonstrated, in
FIGS. 4A and B, that the BtPGSI208 and BtPGSI245 protoxins and
toxins are immunologically related to the Bt13 toxin. In addition,
the BtPGSI245 protoxin was also shown, in FIG. 4B, to be
immunologically related to the Bt2 protoxin. After blotting, the
proteins were stained with Indian ink (Sutherland and Skerritt,
1986) to show both immunoreactive and non-immunoreactive proteins
(FIGS. 4A and 4B).
EXAMPLE 3
Insecticidal activity of the BtPGSI208 and BtPGSI245 crystal
proteins
As in Example 2, both strains were grown for 48 to 72 hrs at
28.degree. C. on T.sub.3 medium. After sporulation, the spores and
crystals were harvested in PBS (phosphate buffered saline) with a
Trihalski spatula. The resulting spore-crystal suspensions were
centrifuged, and the pellets were resuspended and incubated
overnight in aqueous Na.sub.2 CO.sub.3 and DTT solutions as
described in Example 2. After centrifugation, the supernatants were
recovered, and their contents of the respective crystal proteins of
the two strains were determined.
Potato leaves were dipped in aqueous dilutions of the supernatant
solutions and then air dried for two hours. Colorado potato beetle
larvae of the second instar were placed on the treated leaves, and
mortality of the larvae was measured after three days. These
results were compared with the mortality of larvae fed leaves
treated with solubilized crystal proteins of Bt HD-1 ("Bt kurstaki
Dipel" from Abbott Laboratories, Abbott Park, North Chicago, Ill.,
U.S.A.) as a control. LC.sub.50, expressed as ug of solubilized
crystal proteins/ml, was calculated by Probit analysis (Finney,
1971). The results are summarized in Table 5, below.
TABLE 5 ______________________________________ Comparison of
toxicity of solubiliized crystal proteins from the BtPGSI208
strain, the BtPGSI245 strain and the BtHD1 strain (control) against
larvae of Leptinotarsa decemlineata. Strain LC50 FL95min FL95max
Slope ______________________________________ BtPGSI208 5.0 3.5 7.3
2.4 BtPGSI245 25.1 14.7 43.3 1.5 Control >500 -- -- --
______________________________________
EXAMPLE 4
Identification and cloning of the btPGSI208 gene
The BtPGSI208 protoxin from the BtPGSI208 strain was detected by
ELISA (Engvall and Pesce, 1978) with a polyclonal antiserum against
the Bt13 coleoptera toxin (Hofte et al, 1987). The btPGSI208 gene
was identified in the BtPGSI208 strain by preparing total DNA of
the BtPGSI208 strain and then digesting the DNA with the
restriction enzymes HindIII, EcoRI and ClaI. The so-digested DNA
was analyzed by Southern blotting, probing with a nick-translated
2.9 kb HindIII fragment from the genome of the BtS1 strain
(European patent application 88/402,115.5) containing the bt13
gene. After hybridization with the probe, the blot was washed under
low stringency conditions (2XSSC, 0.1%SDS at 68.degree. C. for
2.times.15 min), showing the presence of the btPGSI208 gene,
related to the bt13 gene. The hybridization pattern with the probe
also showed that the btPGSI208 gene was clearly different from the
bt13 gene.
In order to isolate the btPGSI208 gene, total DNA was prepared from
the BtPGSI208 strain. The total DNA preparation was partially
digested with Sau3A and was size-fractionated on a sucrose
gradient. Fractions containing DNA between 5 kb and 10 kb were
ligated to the BglII-digested and bovine alkaline phosphatase
("BAP")-treated cloning vector pEcoR251 (Deposited at the DSM on
Jul. 13, 1988 under accession number DSM 4711). Recombinant E. coli
clones containing the vector were then screened with the 2.9 kb
HindIII DNA fragment containing the bt13 gene, as a probe, to
identify DNA fragments of clones containing the btPGSI208 gene. The
so-identified DNA fragments were then sequenced (FIG. 1) according
to Maxam and Gilbert (1980).
Based on the analysis of the DNA sequence of the btPGSI208 gene,
the gene is cut with an appropriate restriction enzyme to give the
truncated btPGSI208 gene, encoding the BtPGSI208 toxin of about 68
kDa.
EXAMPLE 5
Identification and cloning of the btPGSI245 gene
The BtPGSI245 protoxin from the BtPGSI245 strain was detected by
ELISA (Engvall and Pesce, 1978) with a polyclonal antiserum
directed against the Bt13 coleoptera toxin. Colony hybridization of
the BtPGSI245 strain and Southern blotting of the BtPGSI245 total
DNA, that had been probed with the 2.9 kb HindIII DNA fragment
containing the bt13 gene and washed under the previously described
low stringency conditions, revealed no hybridizing DNA.
In order to isolate the btPGSI245 gene, total DNA from the
BtPGSI245 strain was prepared and partially digested with Sau3A.
The digested DNA was size fractionated on a sucrose gradient and
fragments ranging from 5 kb to 10 kb were ligated to the
BamHI-digested and BAP-treated cloning vector pUC18
(Yannisch-Perron et al, 1985). Recombinant clones containing the
vector were then screened by colony immunoprobing (French et al,
1986) with an antiserum against the Bt13 toxin. Positive colonies
were purified, and total protein preparations were again analyzed
by immunoblotting with the antiserum against the Bt13 toxin. DNA
fragments, containing the BtPGSI245 gene, from clones expressing
the BtPGSI 245 protoxin, were then sequenced (FIG. 2) according to
Maxam and Gilbert (1980).
Based on the analysis of the DNA sequence of the btPGSI245 gene,
the gene is cut with an appropriate restriction enzyme to give the
truncated btPGSI245 gene, encoding the BtPGSI245 toxin of about 66
kDa.
EXAMPLE 6
Construction of a btPGSI208-neo hybrid gene and a btPGSI245-neo
hybrid gene
Following the procedure of U.S. patent application Ser. No. 821,582
and European patent applications 88/402,115.5 and 86/300291.1, the
truncated btPGSI208 and btPGSI245 genes from Examples 4 and 5,
respectively, are each fused to the neo gene to form the
corresponding hybrid gene.
EXAMPLE 7
Insertion of the btPGSI208 and btPGSI245 genes, the truncated
btPGSI208 add btPGSI245 genes and the btPGSI208-neo and
btPGSI245-neo hybrid genes in E. coli and insertion of the
truncated btPGSI208 and btPGSI245 genes and the btPGSI208-neo and
btPGSI245-neo hybrid genes in potato plants
The btPGSI208 and btPGSI245 genes and the truncated btPGSI208 and
btPGSI245 genes from Examples 4 and 5 and the btPGSI208-neo and
btPGSI245-neo hybrid genes from Example 6 are each inserted into,
and expressed by, different E. coli in a conventional manner, using
E. coli expression vectors as described by Botterman and Zabeau
(1987). In order to express these genes in E. coli and in plants,
different gene cassettes are made. In this regard, a BamHI
restriction site is introduced at the ATG initiation codon of the
btPGSI208 gene by site directed mutagenesis (Stanssens et al, 1988;
Stanssens et al, 1989). The 4th nucleotide of the btPGSI208 gene is
changed from an A to a G, yielding a unique BamHI site. According
to the "Kozak rules" (Kozak, 1986), this mutation should also
optimize the translation initiation in plant cells. In this way,
the AAT codon (second codon) coding for Asn is changed into a GAT
codon coding for Asp. Similarly, a NcoI is introduced at the ATG
translation initiation codon of the btPGSI245 gene.
The insecticidal activity, against Colorado potato beetle second
instar larvae, of the BtPGSI208 protoxin produced in E. coli
(transformed with the btPGSI208 gene) is given in Table 6, below.
Toxicity is expressed as 50% lethal concentration in ug/ml,
followed by the 95% confidence interval and the slope of the probit
line.
TABLE 6 ______________________________________ LC50 (ug/ml) slope
______________________________________ 1.0 (0.6-1.8) 2.3
______________________________________
The toxicity of the BtPGSI208 protoxin produced in E. coli was
about 5 times greater than the toxicity of the BtPGSI208 protoxin
produced by the BtPGSI208 strain. Trypsin digestion of the
BtPGSI208 protoxin, produced in E. coli, yielded the BtPGSI208
toxin which also was active against Colorado potato beetle.
Using the procedures described in U.S. patent application Ser. No.
821,582 and European patent applications 86/300,291.1 and
88/402,115.5, the truncated btPGSI208 and btPGSI245 genes and the
btPGSI208-neo and btPGSI245-neo hybrid genes are isolated and are
cloned into the intermediate T-DNA vector, pGSH160 (Deblaere et al,
1988), between the vector's T-DNA terminal border repeat sequences.
To provide major expression in plants, the hybrid genes and
truncated genes are placed under the control of the strong
constitutive promoters, Cabb-JI 35S promotor (Hull and Howell,
1987) or the TR2' promoter (Velten et al, 1984), and are fused to
the transcription termination and polyadenylation signals of the
octopine synthase gene (Gielen et al, 1984).
Using standard procedures (Deblaere et al, 1985), the intermediate
plant expression vectors, containing the truncated btPGSI208 and
btPGSI245 genes and the btPGSI208-neo and btPGSI245-neo hybrid
genes, are transferred into the Agrobacterium strain C 58 C1
Rif.sup.R (US patent application Ser. No. 821,582; European patent
application 86/300,291.1) carrying the disarmed Ti-plasmid pGV2260
(Vaeck et al, 1987). Selection for spectinomycin resistance yields
cointegrated plasmids, consisting of pGV2260 and the respective
intermediate plant expression vectors. Each of these recombinant
Agrobacterium strains is then used to transform different potato
plants (Solanum tuberosum) so that the truncated btPGSI208 gene,
the truncated btPGSI245 gene, the btPGSI208-neo hybrid gene and the
btPGSI245-neo hybrid gene are contained in, and expressed by,
different potato plant cells.
EXAMPLE 8
Expression of the truncated btPGSI208 and btPGSI245 genes and the
btPGSI208-neo and btPGSI245-neo hybrid genes in potato plants
The insecticidal activity against Coleoptera of the expression
products of the truncated btPGSI208 and btPGSI245 genes and the
btPGSI208-neo and btPGSI245-neo hybrid genes in leaves of
transformed potato plants, generated from the transformed potato
plant cells of Example 7, is evaluated by recording the growth rate
and mortality of Leptinotarsa decemlineata larvae fed on these
leaves. These results are compared with the growth rate of larvae
fed leaves from untransformed potato plants. Toxicity assays are
performed as described in European Patent application 88/402,115.5,
U.S. patent application 821,582 and European patent application
86/300,291.1. A significantly higher mortality rate is obtained
among larvae fed on leaves of transformed potato plants containing
the truncated btPGSI208 gene, the truncated btPGSI245 gene, the
btPGSI208-neo hybrid gene or the btPGSI245-neo hybrid gene than
among larvae fed the leaves of untransformed plants.
EXAMPLE 9
Transformation of a Bt strain with the endogenous btPGSI208
gene
In order to enhance its insecticidal spectrum, the BtS174A strain
(Mahillon et al, 1989) is electroporated with plasmid pAMbt21R1.
pAMbt21R1 is obtained by cloning the 5.4 kb HpaI fragment of
plasmid pJL21 into the EcoRV site of the tetracycline resistance
gene of the shuttle vector pAM401 (Wirth et al, 1987). Plasmid
pJL21 is obtained by cloning a 6 kb fragment, containing the
btPGSI208 gene, from the BtPGSI208 strain into the BglIII site of
plasmid pEcoR251 (DSM accession no. 4711).
The electroporation procedure is carried out as described in PCT
patent application no. PCT/EP89/01539. The BtS174A strain,
transformed with pAMbt21R1, retains its insecticidal activity
against Lepidoptera and demonstrates a newly acquired insecticidal
activity against Coleoptera due to expression of the btPGSI208 gene
in the strain.
Needless to say, this invention is not limited to the BtPGSI208
(DSM 5131) strain and the BtPGSI245 (DSM 5132) strain. Rather, the
invention also includes any mutant or variant of the BtPGSI208 or
BtPGSI245 strain which produces crystals, crystal proteins,
protoxin or toxin having substantially the same properties as the
BtPGSI208 or BtPGSI245 crystals, crystal proteins, protoxin or
toxin. In this regard, variants of the BtPGSI208 and BtPGSI245
strains include variants whose total protein pattern is
substantially the same as the protein pattern of either the
BtPGSI208 strain or the BtPGSI245 strain as shown in FIG. 3.
This invention also is not limited to potato plants transformed
with the truncated btPGSI208 or btPGSI245 gene. It includes any
plant, such as tomato, tobacco, rapeseed, alfalfa, sunflowers,
cotton, corn, soybeans, brassicas, sugar beets and other
vegetables, transformed with an insecticidally effective part of
the btPGSI208 or btPGSI245 gene.
Nor is this invention limited to the use of Agrobacterium
tumefaciens Ti-plasmids for transforming plant cells with an
insecticidally effective btPGSI208 or btPGSI245 gene part. Other
known techniques for plant cell transformations, such as by means
of liposomes, by electroporation or by vector systems based on
plant viruses or pollen, can be used for transforming
monocotyledons and dicotyledons with such a gene part.
Furthermore, DNA sequences other than those shown in FIG. 1 for the
btPGSI208 gene and the truncated btPGSI208 gene and in FIG. 2 for
the btPGSI245 gene and the truncated btPGSI245 gene can be used for
transforming plants and bacteria. In this regard, the DNA sequence
of FIG. 1 or 2 can be modified by: 1) replacing some codons with
others that code either for the same amino acids or for other amino
acids; and/or 2) deleting or adding some codons; provided that such
modifications do not substantially alter the properties of the
encoded, insecticidally effective portion of the BtPGSI208 or
BtPGSI245 protoxin or toxin.
Also, other DNA recombinants containing the aforementioned DNA
sequences in association with other foreign DNA, particularly the
DNA of vectors suitable for transforming plants and microorganisms
other than E. coli, are encompassed by this invention. In this
regard, this invention is not limited to the specific plasmids
containing the btPGSI208 and btPGSI245 genes, or parts thereof,
that were heretofore described, but rather, this invention
encompasses any DNA recombinants containing a DNA sequence that is
their equivalent. Further, the invention relates to all DNA
recombinants that include all or part of either the btPGSI208 gene
or the btPGSI245 gene and that are suitable for transforming
microorganisms (e.g., plant associated bacteria such as Bacillus
subtilis, Pseudomonas and Xanthomonas or yeasts such as
Streptomyces cerevisiae) under conditions which enable all or part
of the gene to be expressed and to be recoverable from said
microorganisms or to be transferred to a plant cell.
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